Experiment 1 used one pea cultivar (2.94 mg trypsin inhibitor units (TIU /g DM) processed under two extrusion temperatures (exit temperatures of 70 and 140 °C) and two moisture levels (0 and 1.96l water added/h) giving a 2 × 2 factorial design (with a 5th treatment being the raw pea sample).
One calpastatin molecule contains four inhibitor units; each unit inhibits one calpain molecule with variable efficiency.
Inhibitor activity was calculated by the amount of purified sample required to inhibit 50% of trypsin activity, which is considered as one unit of trypsin inhibition and expressed as trypsin inhibitor units per mg seed protein.
After intracerebroventricular administration (30 min), animals were killed by decapitation, and plasma was collected in plastic lithium heparin tubes containing 4,200 kallikrein inhibitor units (KIU) aprotinin (Bayer; Haywards, Heath, UK).
Blood samples for analysis of adipokines and CRP were distributed in iced tubes containing 1.5 mg EDTA, and for analysis of insulin, the tubes contained 500 Kallikrein inhibitor units Trasylol and 15 mg of EDTA per mL of blood.
A volume of 20 mL of blood was collected in a Sterilin tube containing 330 μL of Trasylol (= 3000 KIU (Kallikrein Inhibitor Units)) and 80 μL of 1 M EDTA per 20 mL of blood.
Blood samples were collected at t = −60, 0, 15, 30, 45, 60, 75, 90, and 120 min into lithium heparin-coated tubes containing 1500 kallikrein inhibitor units (0.15 ml) aprotinin (Trasylol, Bayer Schering Pharma, Berlin).
Blood samples for the determination of Ang-(1 7) were collected into tubes containing 20 μl of an inhibitor cocktail [50 mol/l Na2EDTA, 0.2 mol/l N-ethylmaleimide and 1 2 TIU (trypsin inhibitor units)/ml aprotinin made up in saline] per ml of blood.
Cells were washed with PBS and lysed in TOTEX buffer (20 mM HEPES [pH 7.9], 0.35 mM NaCl, 20%% glycerol, 1 % Nonidet P-40, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 50 μM PMSF and 90 trypsin inhibitor units [TIU's]/ml aprotinin) for 30 minutes on ice.
Cells cultured in 6-well plate dishes were lysed for 30 min in 0.3 ml of 1% Triton X-100 lysis buffer (20 mM Tris-HCL [pH 7.5], 150 mM NaCl, and 1% Triton X-100) containing 5 μg ml-1 lupeptin, 50 mM phenylmethylsulfonyl fluoride, and 7.2 trypsin inhibitor units for aprotinin.
